• Gaia Colasante, Gabriele Lignani, Simone Brusco, Claudia Di Berardino, Jenna Carpenter, Serena Giannelli, Nicholas Valassina, Simone Bido, Raffaele Ricci, Valerio Castoldi, Silvia Marenna, Timothy Church, Luca Massimino, Giuseppe Morabito, Fabio Benfenati, Stephanie Schorge, Letizia Leocani, Dimitri M Kullmann, and Vania Broccoli.
• Stem Cell and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy. Electronic address: colasante.gaia@hsr.it.
• Mol. Ther. 2020 Jan 8; 28 (1): 235-253.

AbstractDravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

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